RNA and DNA analysis at the single cell level is crucial to the understanding of the heterogeneity within cell populations, and new tools for this work are just emerging (Kalisky et al. (2011) Annu. Rev. Genet. 45:431-445). Recent progress in microfluidics has revolutionized the area and created new capabilities for single cell analysis (Zare et al. (2010) Annu. Rev. Biomed. Eng. 12:187-201). For example, Marcus et al. (Analytical Chemistry (2006) 78:956-958) demonstrated an integrated microfluidic chip that performed single cell lysis, RNA purification, and complementary DNA (cDNA) synthesis. Since then, the technique has been improved (White et al. (2011) Proc. Natl. Acad. Sci. USA 108:13999-14004), and similar techniques have been used to measure single cells (thong et al. (2008) Lab on a Chip 8:68-74). The technique is now well established but requires a specialized system (e.g., pumping liquids and valving). Further, the basic idea of quantifying the amount of RNA has relied upon its conversion to cDNA and subsequent amplification by enzymatic processes such as quantitative polymerase chain reaction (qPCR). Such basic approaches are effective, but may not be optimal, as PCR is well-known to introduce sequence-specific bias (Kalisky et al., supra). Because of this, most findings require validation by in situ hybridization or staining.
Capillary electrophoresis (CE) methods using either traditional free-standing capillaries or on-chip CE have also been used for handling and analyzing molecules from single cells (Borland et al. (2008) Annu. Rev. Anal. Chem. 1:191-227). However, few studies have focused on direct detection of RNA without amplification. Han and Lillard (Anal. Chem. (2000) 72:4073-4079) demonstrated direct measurement of RNA from a single cell and obtained an electropherogram of ribosomal RNA. They performed cell lysis inside the same capillary used for separation using sodium dodecyl sulfate (SDS). Their protocol separated RNA by CE and quantified RNA using an ethidium bromide label and laser-induced fluorescence detection. In subsequent work, Lillard's group examined RNA expression in various phases of the cell cycle (G1, S, G2, and M) and reported changes of total amount of RNA and individual RNA sequences over each phase (Han & Lillard (2002) Anal. Biochem. 302:136-143). Their limit of detection for CE was well below the single cell level. However, their protocol provided only the relative amount of the RNA, and no simultaneous RNA and DNA information.
Thus, there remains a need for better methods of extracting and measuring total cytoplasmic RNA and nuclear DNA from single cells.